CN100359008C - Reagent for determining analyte concentration of patient by enzyme method - Google Patents
Reagent for determining analyte concentration of patient by enzyme method Download PDFInfo
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- CN100359008C CN100359008C CNB038269554A CN03826955A CN100359008C CN 100359008 C CN100359008 C CN 100359008C CN B038269554 A CNB038269554 A CN B038269554A CN 03826955 A CN03826955 A CN 03826955A CN 100359008 C CN100359008 C CN 100359008C
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Abstract
The present invention discloses a reagent for determining the analyte concentration of a patient by a nenzyme method. At the time of detection, the oxidation rate of reduced coenzyme in the reagent is measured, and the present invention comprises a coenzyme reduction system which causes the reduced coenzyme to be continuously regenerated in the storage period of the reagent to realize secular stability of the reagent. An enzyme /substrate pair used in the coenzyme reduction system has complete specificity, and accordingly, the dosages of enzyme and a substrate are reduced; the reagent stability is increased, and the reagent is a liquid single reagent. The present invention also discloses a reagent for measuring aspartate aminotransferase, alanine aminotransferase and carbamide.
Description
Technical field
The present invention relates to the reagent that enzymatic assays patient analyte concentration is used, particularly relate to the reagent of using with in the method for quantitatively determining that is present in reduced coenzyme oxidation in the directly related reaction sample of analyte concentration in the sample.
Background technology
Clinically, (β-NADH) is oxidized to nadide (β-NAD to use DPNH
+) the analyte measured of detection technique comprise: aspartic transaminase (AST); Alanine aminotransferase (ALT), urea (UREA), serum lactic dehydrogenase (LDH-P), AHB (α-HBDH), ammonia (NH
3) and carbonic acid gas (CO
2) etc.
Aspartic transaminase (AST) is distributed widely in human body, and higher concentration is arranged in the heart, liver, skeletal muscle, kidney and red corpuscle.The damage of these tissues, as myocardial infarction, viral hepatitis, hepatic necrosis, diseases such as liver cirrhosis and malnutrition all can cause the rising of AST level in serum or the blood plasma.
When measuring the AST activity, the conversion of the amino of AST catalysis α-Tong Wuersuan and L-aspartic acid forms L-L-glutamic acid and oxaloacetic acid in the serum.(β-NADH) and in the presence of the malate dehydrogenase (malic acid dehydrogenase) (MDH), oxaloacetic acid is reduced to oxysuccinic acid, and β-NADH is oxidized to nadide (β-NAD at DPNH
+), thereby cause that absorbancy descends.The speed of the decline of this absorbancy is directly proportional with the AST activity, so can measure the AST activity with the spectrophotometry monitoring at the β of 340nm place-NADH absorbancy fall off rate
The serum lactic dehydrogenase that exists in the serum can make the endogenous pyruvic acid be converted into lactic acid, and simultaneous oxidation β-NADH and distrubed test add high density serum lactic dehydrogenase (LDH), can eliminate the interference of endogenous pyruvic acid in lag period fast.
Alanine aminotransferase (ALT) has higher concentration in liver, then contain less in kidney, the heart, skeletal muscle, lungs.Usually the rising of ALT is to be caused by some and liver diseases associated, comprises liver cirrhosis, liver cancer, viral or toxic hepatitis and obstructive jaundice etc.
When measuring the ALT activity, the amino conversion of serum alt catalysis α-Tong Wuersuan and L-L-Ala forms L-L-glutamic acid and pyruvic acid, and in the presence of β-NADH and serum lactic dehydrogenase (LDH), pyruvic acid is reduced to L-lactic acid, and β-NADH is oxidized to β-NAD
+Thereby, cause that absorbancy descends.The fall off rate of this absorbancy is directly proportional with the ALT activity, so can measure the ALT activity with the spectrophotometry monitoring at the β of 340nm place-NADH absorbancy fall off rate.
The interference of endogenous pyruvic acid in the serum can be eliminated in lag period fast by adding excessive serum lactic dehydrogenase (LDH).
Urea (UREA) is the metabolic primary product of human body internal protein, it has constituted the non-protein nitrogen(NPN) of the overwhelming majority in the blood, urea in liver, produce and by renal excretion to urine in, the infraction of various ephrosis and urinary tract can cause that all blood urea raises, so blood urea is the leading indicator of renal function.
When measuring urea, urea is decomposed into ammonia and carbonic acid gas under the catalysis of urase, and ammonia and α-Tong Wuersuan form L-glutamic acid in the presence of β-NADH and glutamate dehydrogenase (GLDH), and β-NADH is oxidized to β-NAD simultaneously
+Thereby, cause the decline of absorbancy, be directly proportional with content of urea in the sample at the fall off rate of 340nm place absorbancy, so can record content of urea at the β of 340nm place-NADH absorbancy fall off rate with the spectrophotometry monitoring.
Endogenous ammonia disturbs and can eliminate in lag period in the sample.
AST, ALT, UREA detection reagent will be mixed with liquid single reagent steady in a long-term, key is to solve toolenzyme and the DPNH (stability problem of β-NADH).Because enzyme is the meticulous protein of a kind of structure, stable extreme difference, temperature, pH, ionic strength, impurity, metal ion, microorganism etc. all can influence its activity.Improve the stability of enzyme in the aqueous solution, can adopt the condition of optimizing the environment, add stablizer and sanitas etc.Toolenzyme should select for use contain that assorted enzyme is few, Heat stability is good, the enzyme of pH stable range in test pH scope.The consumption of toolenzyme is suitable, should guarantee that it has quite long stationary phase in liquid reagent, guarantee that again test result is accurate.
The difficult point of guaranteed reagent,G.R. stability mainly is DPNH, and (β's-NADH) is stable, and (β-NADH) is the common indicator that AST, ALT, three kinds of reagent of UREA detect to DPNH.For guaranteed reagent,G.R. reaches due linearity, β-NADH should keep certain concentration in the reagent, promptly can not be lower than 1.0A in 340nm place absorbancy.But β-NADH is unsettled in the aqueous solution of pH＜8.6, can spontaneous oxidation be β-NAD
+, also can be subjected to the catalysis of various assorted enzymes in the solution, be oxidized to β-NAD
+
In order to increase the stable of β-NADH, as far back as the seventies in last century just the someone done a large amount of research work, except using general physical method as reagent being made dried frozen aquatic products or dry powder, or with outside the increase NADH stability such as anhydrous organic solvent, Modrovich in 1977 is at his patent (US Patent 4,394,449) proposed in glucose-6-phosphate dehydrogenase (G6PD) (G-6-PDH)/G-6-P (G-6-P) unstable product β-NAD of β-NADH
+Again backward reaction generates β-NADH, plays the purpose of dynamic stability β-NADH.
But because enzyme engineering does not at that time also develop into the level of today, the technical problem of essence does not solve, and they can only make double reagent, and the stability that is merged into the liquid single reagent has only one month to three months.The principle of 90 years F.Hoffmann La RocheAG (AU-A-61906/90) utilization Modrivich Ivan E is done a lot again, with unstable product β-NAD
+Again become β-NADH.But his method also can only be made into double reagent, in case be made into single reagent, stability is just very poor.The shortcoming of the similar approach that people such as Klose propose in patent US Pantent 4,019,916 is that the test duration is long, and only is applicable to that contain can be by the reagent system of phosphorylated substrate.Most representative, with this subject in close relations be that Australian J. De Qiaojiao equals 1996.2.26 at Chinese patents (CN 1179792A), it uses non-specificity enzyme/substrate right, successfully the dynamic stability technology has been applied in the double reagent of the single reagent of AST, ALT and UREA, can be with the prolonged-stability of AST and ALT liquid single reagent to the 6-8 month.But stipulated coenzyme reduction system " enzyme has incomplete specificity to substrate " in patent, " enzyme/substrate is to being glucose-6-phosphate dehydrogenase (G6PD)/D-glucose " is though J. De Qiaojiao on forefathers' basis, has obtained new achievement again.But glucose-6-phosphate dehydrogenase (G6PD) that is to use and D-glucose amount are all very big, and the enzyme amount is 3500U/L, and the D-glucose amount is 18.016g/L.So not only obviously increase the cost of reagent, but also might introduce new assorted enzyme.
Summary of the invention
The objective of the invention is to above-mentioned deficiency, propose the reagent that a kind of enzymatic assays patient analyte concentration is used, wherein the rate of oxidation of reduced coenzyme is measured at prior art.It is the cost of not obvious increase reagent also, can prevent assorted enzyme introducing, has permanent stability preferably.
For this reason, the reagent that provides a kind of enzymatic assays patient analyte concentration to use, during mensuration the rate of oxidation of reduced coenzyme in the reagent is measured, described reagent is realized the long-term stability of reagent in the dynamic stability effect of this reagent lay up period cyclic regeneration reduced coenzyme by the right coenzyme reduction system of certain enzyme/substrate, described enzyme and substrate centering, enzyme has specificity completely to substrate.
Described reagent is the liquid single reagent; Enzyme/substrate is to the preferred Hexose phosphate dehydrogenase/D-glucose of using in the described coenzyme reduction system.
The reagent that the present invention also provides enzymatic assays patient's aspartic transaminase concentration to use, during mensuration the rate of oxidation of reduced coenzyme in the reagent is measured, described reagent is realized the long-term stability of reagent by the right coenzyme reduction system of certain enzyme/substrate in the dynamic stability effect of this reagent lay up period cyclic regeneration reduced coenzyme, and this substrate is had completely specificity to this enzyme and described reagent is the liquid single reagent.Described enzyme/substrate is to the preferred Hexose phosphate dehydrogenase/D-glucose of using.Described Hexose phosphate dehydrogenase and D-glucose consumption are selected 2-100U/L and 0.1-20mmol/L respectively for use, preferably use 5-50U/L and 1-10mmol/L.
The reagent that the present invention also provides enzymatic assays patient's alanine aminotransferase concentration to use, during mensuration the rate of oxidation of reduced coenzyme in the reagent is measured, described reagent is realized the long-term stability of reagent by the right coenzyme reduction system of certain enzyme/substrate in the dynamic stability effect of this reagent lay up period cyclic regeneration reduced coenzyme, and this substrate is had completely specificity to this enzyme and described reagent is the liquid single reagent.Described enzyme/substrate is to the preferred Hexose phosphate dehydrogenase/D-glucose of using.Described Hexose phosphate dehydrogenase and D-glucose consumption are selected 2-100U/L and 0.1-20mmol/L respectively for use, preferably use 2-50U/L and 1-10mmol/L.
The reagent that the present invention also provides enzymatic assays patients'blood urea concentration to use, during mensuration the rate of oxidation of reduced coenzyme in the reagent is measured, described reagent is realized the long-term stability of reagent by the right coenzyme reduction system of certain enzyme/substrate in the dynamic stability effect of this reagent lay up period cyclic regeneration reduced coenzyme, and this substrate is had completely specificity to this enzyme and described reagent is the liquid single reagent.Described enzyme/substrate is to the preferred Hexose phosphate dehydrogenase/D-glucose of using.Described Hexose phosphate dehydrogenase and D-glucose consumption are selected 2-100U/L and 0.1-20mmol/L respectively for use, preferably use 5-50U/L and 1-10mmol/L.
In using desaturase/substrate regeneration reducing β-NADH system, we select Hexose phosphate dehydrogenase and its 100% specificity substrate D-glucose for use.D-glucose is oxidized to maltonic acid lactones, β-NAD
+Be reduced to β-NADH.
The pH stable range of Hexose phosphate dehydrogenase is 6-8.5, is stable in reagent test PH 7.5-8.2 scope.The optimal pH of Hexose phosphate dehydrogenase is 8.0, also in reagent test pH7.5-8.2 scope.Enzymic catalytic reaction speed is the highest, in the β-NADH restoring system of optimal pH, can reduce desaturase and substrate consumption, prevents to introduce new assorted enzyme and influences reagent stability, and increase reagent cost hardly.
β-NADH reducing/regenerating speed of reaction can control with Hexose phosphate dehydrogenase and D-glucose add-on, and it is suitable generally to control regenerative response speed and β-NADH natural oxidation speed.Like this, the coenzyme reducing/regenerating system of Hexose phosphate dehydrogenase/D-glucose stores continuous reproducible β-NADH in period at reagent, and will can not influence test result when reagent test.
The Hexose phosphate dehydrogenase consumption that is used for β-NADH reducing/regenerating system is selected 2-100U/L for use, the D-glucose concn is selected 0.1-20mmol/L for use, too high Hexose phosphate dehydrogenase and D-glucose consumption can make β-NADH reducing/regenerating excessive velocities, produce negative interference when reagent detects.
Described in the beginning of this specification sheets, for be used for measuring patient AST content, reagent of the present invention except that Hexose phosphate dehydrogenase/D-glucose as the coenzyme reduction system, other materials that also need have: malate dehydrogenase (malic acid dehydrogenase) (MDH), serum lactic dehydrogenase (LDH), DPNH (β-NADH), L-aspartic acid and α-Tong Wuersuan.
For be used for measuring patient ALT content, reagent of the present invention except that Hexose phosphate dehydrogenase/D-glucose as the coenzyme reduction system, other materials that also need have: serum lactic dehydrogenase (LDH), DPNH (β-NADH), L-L-Ala and α-Tong Wuersuan.
For be used for measuring patient UREA content, reagent of the present invention except that Hexose phosphate dehydrogenase/D-glucose as the coenzyme reduction system, other materials that also need have: urase, glutamate dehydrogenase (GLDH), DPNH (β-NADH) and α-Tong Wuersuan.
Reagent of the present invention can also comprise buffer reagent, sanitas, stablizer, sequestrant etc. and has the effect of enhanced stability and do not influence other compositions of characteristic of the present invention in fact except comprising the required coenzyme reduction system of determination and analysis substrate concentration and other basic substrate and enzyme.
Glycerine, sugar and ethylene glycol are polyols, can form a lot of hydrogen bonds with protein molecule, and help to form " solvent layer ", and the solvent layer around this kind of enzyme molecule is different with whole water, and they can increase surface tension and soltion viscosity.This class additive is by to proteinic effective dehydration, and reduce protein hydrolysis and play stabilized enzyme, so can be with relative low molecular weight polyols stabilized enzyme.We select glycerine or the ethylene glycol stablizer as enzyme for use, and the glycerine of excessive concentrations increases soltion viscosity, are unfavorable for test.
EDTA disodium salt and heavy metal ion form coordination complex, prevent the inhibition of heavy metal ion to enzymic activity.
The microbial contamination meeting reduces the stability of enzyme, adds sanitas and can suppress microorganism growth.Preferred sanitas is a sodium azide among the present invention.
Among the present invention, the liquid single reagent AST that using glucose desaturase/D-glucose is stablized the stabilization technology preparation of β-NADH consists essentially of:
Coenzyme reduction system (Hexose phosphate dehydrogenase/D-glucose), L-aspartic acid, α-Tong Wuersuan, malate dehydrogenase (malic acid dehydrogenase) (MI) H), serum lactic dehydrogenase (LDH), DPNH (β-NADH).
Preferably include in addition: Tris-HCl damping fluid, potassium hydroxide, EDTA disodium salt, glycerine, sodium azide.
Wherein the concentration of Tris-HCl damping fluid is selected 20-100mmol/L for use; α-Tong Wuersuan concentration is selected 6-18mmol/L for use, and α-Tong Wuersuan has absorption at 340nm, and concentration should not be too high; L-aspartic acid concentration is selected 100-300mmol/L for use; The adding of potassium hydroxide is in order to help L-aspartic acid dissolving, consumption and L-aspartic acid for etc. mole; EDTA disodium salt and metal ion form coordination complex, prevent the activity of heavy metal ion inhibitory enzyme, and concentration is selected for use and is 1-10mmol/L; The concentration of β-NADH is selected 0.1-0.3mmol/L for use, and linearity range diminished when AST tested when being lower than 0.1mmol/L, influenced test result, was higher than 0.3mmol/L and can makes the reagent blank absorbancy too high; The malate dehydrogenase enzyme dosage is selected 100-2500U/L for use; The adding of serum lactic dehydrogenase can be eliminated the interference of endogenous pyruvic acid in the sample, and the lactic dehydrogenase enzyme dosage is selected 1000-4000U/L for use; The adding of Hexose phosphate dehydrogenase/D-glucose is the unstable product β-NAD that makes β-NADH
+Again be regenerated as β-NADH, relatively stable with β-NADH in the guaranteed reagent,G.R., the Hexose phosphate dehydrogenase consumption is selected 2-100U/L for use; The D-glucose concn is selected 0.1-20mmol/l for use; Glycerine has stabilization to enzyme, and consumption is selected 1%-20% for use, and high density glycerine can increase soltion viscosity; For preventing microbial contamination, add sodium azide in the reagent as sanitas, consumption is selected 0.1-1.0g/L for use.
A kind of preferred AST reagent according to the present invention's preparation is formulated as follows:
Table 1
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 80-100 | 9.69-12.1g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 12-15 | 2.71-3.39g |
| The L-aspartic acid | 133.1 | 200-240 | 26.6-31.9g |
| Potassium hydroxide | 56.1 | 200-240 | 11.2-13.5g |
| D-glucose | 180.2 | 1-10 | 0.18-1.8g |
| Glycerine | 92.1 | 5％-10％ | |
| EDTA.2Na | 372.2 | 3-5 | 1.12-1.86g |
| Sodium azide | 65.1 | 0.3-0.5g | |
| β-NADH disodium salt | 709.4 | 0.25-0.28 | 0.18-0.2g |
| Serum lactic dehydrogenase | 1500-2000U | ||
| Malate dehydrogenase (malic acid dehydrogenase) | 800-1000U | ||
| Hexose phosphate dehydrogenase | 5-50U | ||
| Hydrochloric acid | 36.5 | Transfer pH7.8-8.1 |
Among the present invention, the liquid single reagent ALT that using glucose desaturase/D-glucose is stablized the stabilization technology preparation of β-NADH consists essentially of:
Coenzyme reduction system (Hexose phosphate dehydrogenase/D-glucose), L-L-Ala, α-Tong Wuersuan, serum lactic dehydrogenase (LDH), DPNH (β-NADH).
Preferably include in addition: Tris-HCl damping fluid, EDTA disodium salt, glycerine, sodium azide.
Wherein the Tris-HCl buffer concentration is selected 20-100mmol/L for use; α-Tong Wuersuan concentration is selected 8-18mmol/L for use; L-L-Ala concentration is preferably used 200-800mmol/L; The EDTA disodium salt is selected 1-10mmol/L for use; β-NADH consumption is selected 0.1-0.30mmol/L for use; The consumption of serum lactic dehydrogenase should guarantee to eliminate fast the interference of endogenous pyruvic acid, also will guarantee the linearity range of catalyzed reaction, selects 1000-4000U/L for use; The Hexose phosphate dehydrogenase consumption is selected 2-100U/L for use; The D-glucose concn is selected 0.1-20mmol/L for use, and the glycerine consumption is selected 1%-20% for use, and the sodium azide consumption is selected 0.1-100g/L for use
A kind of preferred ALT reagent according to the present invention's preparation is as follows:
Table 2
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 80-100 | 9.69-12.1g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 12-15 | 2.71-3.39g |
| The L-L-Ala | 89.1 | 400-500 | 35.6-44.6g |
| D-glucose | 180.2 | 1-10 | 0.72-1.8g |
| The EDTA.2 sodium salt | 372.2 | 3-5 | 1.12-1.86g |
| Glycerine | 92.1 | 5％-10％ | |
| Sodium azide | 65.1 | 0.3-0.5g | |
| β-NADH disodium salt | 709.4 | 0.25-0.28 | 0.18-0.2g |
| Serum lactic dehydrogenase | 3000---4000U | ||
| Hexose phosphate dehydrogenase | 2-50U | ||
| Hydrochloric acid | 36.5 | Transfer pH7.5-7.8 |
Among the present invention, the UREA liquid single reagent that using glucose desaturase/D-glucose is stablized the stabilization technology preparation of β-NADH consists essentially of:
Coenzyme reduction system (Hexose phosphate dehydrogenase/D-glucose), α-Tong Wuersuan, urase, glutamate dehydrogenase (GLDH), DPNH (β-NADH).
Preferably include in addition: Tris-HCl damping fluid, ADP sylvite, glycerine, sodium azide.
Wherein the Tris-HCl buffer concentration is selected 20-150mmol/L for use; α-Tong Wuersuan concentration is selected 1-15mmol/L for use; β-NADH selects 0.1-0.38mmol/L for use; ADP sylvite consumption is selected 0.1-10mmol/L for use; Urase is wanted energy quick catalysis decomposing urea, and consumption is selected 2000-10000U/L for use; Glutamate dehydrogenase may command speed of response adds manyly more, and speed of response is fast more, preferred 200-2000U/L, and the Hexose phosphate dehydrogenase consumption is selected 2-100U/L for use; D-glucose consumption is selected 0.1-20mmol/L for use; The glycerine consumption is selected 1%-30% for use; The sodium azide consumption is selected 0.1-1.0g/L for use.
A kind of preferred UREA reagent according to the present invention's preparation is formulated as follows:
Table 3
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 100-120 | 12.1-14.5g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 4-8 | 0.9-1.8g |
| Sodium azide | 65.0 | 0.2-0.5g | |
| D-glucose | 180.2 | 1-10 | 0.72-1.8g |
| Glycerine | 92.1 | 5％-15％ | |
| ADP.K salt | 501.3 | 1-4 | 0.5-2.0g |
| β-NADH, sodium salt | 709.4 | 0.25-0.3 | 0.18-0.21g |
| Glutamate dehydrogenase | 500-1000U | ||
| Urase | 5000-8000U | ||
| Hexose phosphate dehydrogenase | 5-50U | ||
| Hydrochloric acid | 36.5 | Transfer pH8.0-8.3 |
In above liquid single reagent, serum lactic dehydrogenase should be selected for use has higher affinity to pyruvic acid, does not contain or only contain assorted enzymes such as micro-ALT, GLDH.Malate dehydrogenase (malic acid dehydrogenase) and glutamate dehydrogenase are selected the stability enzyme preferably in the aqueous solution for use.Guaranteeing linear test specification, under time of lag, accuracy and the stable prerequisite, should reduce the consumption of above enzyme, so that reduce the interference of assorted enzyme as far as possible.
The analyte that available reagent of the present invention is measured also comprises except that aspartic transaminase (AST), alanine aminotransferase (ALT), urea (UREA): serum lactic dehydrogenase (LDH-P), AHB (α-HBDH), ammonia (NH
3) and carbonic acid gas (CO
2) etc.
In addition, also can make the DPNH I (coenzyme II (β-NADP of unstable product of β-NADPH) with Hexose phosphate dehydrogenase/D-glucose
+) be reduced to β-NADPH again.
Compared with prior art, the invention has the beneficial effects as follows: used highly narrow spectrum enzyme/substrate right with oxidation resistant coenzyme reduction system owing to be used for stable reagent, the consumption of enzyme and substrate greatly reduces, not only increase reagent cost hardly, and can not introduce new assorted enzyme, thereby improved the stability of reagent because of the adding of a large amount of stabilized enzyme.
Preferred forms
The detailed performance of each side of the present invention will become clearer in the following description of preferred embodiment.
Embodiment 1
The AST reagent that following mensuration is prepared according to the present invention (D-glucose: 5mmol/L, Hexose phosphate dehydrogenase: stability 20U/L): stabilization AST liquid single reagent:
Table 4
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 80 | 9.69g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 12 | 2.71g |
| The L-aspartic acid | 133.1 | 240 | 31.9g |
| Potassium hydroxide | 56.1 | 240 | 13.5g |
| D-glucose | 180.2 | 5 | 0.9g |
| Glycerine | 92.1 | 5％ | |
| EDTA.2Na | 372.2 | 5 | 1.86g |
| Sodium azide | 65.1 | 0.5g | |
| β-NADH disodium salt | 709.4 | 0.27 | 0.19g |
| Serum lactic dehydrogenase | 2000U | ||
| Malate dehydrogenase (malic acid dehydrogenase) | 900U | ||
| Hexose phosphate dehydrogenase | 20U | ||
| Hydrochloric acid | 36.5 | Transfer pH8.0 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization AST liquid single reagent, do not contain glycerine.Other component and concentration thereof and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited.
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 15
Time of lag: 60 second test duration: 60 seconds
The reagent blank absorbancy: the content of reflection β-NADH, initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in quality controlled serum sign value scope
Test is linear: 〉=550U/L
Test result:
1) AST liquid single reagent is deposited the back blank absorbency for 37 ℃
Table 5
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.788 | 1.809 |
| 1 | 1.662 | |
| 2 | 1.538 | |
| 3 | 1.415 | 1.019 |
| 4 | 1.313 | |
| 5 | 1.188 | |
| 6 | 1.148 | |
| 7 | 1.043 |
As seen, stabilization AST liquid single reagent can be deposited 7 days at 37 ℃, and astableization AST liquid single reagent can only be deposited three days.β in the stabilization single reagent-NADH good stability.
2) AST liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 6
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astableization single reagent |
| 0 week | 1.839 | 1.809 |
| 3 weeks | 1.452 | |
| 5 weeks | 1.319 | |
| 7 weeks | 1.210 | |
| 11 weeks | 1.023 | |
| 3 months | 1.665 | |
| 6 months | 1.477 | |
| 9 months | 1.305 | |
| 12 months | 1.102 |
β-NADH can stablize more than 12 months in the 2-8 ℃ of stabilization AST liquid single reagent, but not β-NADH can only stablize 11 weeks (less than three months) in the stabilization AST liquid single reagent.
3) stabilization AST liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 7
| Deposit 3 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 0 | 116 | 233 | 349 | 466 | 582 |
| Measured value U/L | 4.8 | 111 | 227 | 349 | 452 | 585 |
| Deposit 6 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 0 | 113 | 226 | 338 | 451 | 564 |
| Measured value U/L | 5.1 | 115 | 220 | 338 | 436 | 559 |
| Deposit 9 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 0 | 105 | 210 | 315 | 420 | 524 |
| Measured value | 4.9 | 106 | 215 | 315 | 406 | 517 |
| Deposit 13 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 0 | 122 | 244 | 366 | 488 | 610 |
| Measured value | 5.5 | 124 | 247 | 366 | 473 | 582 |
Stabilization AST liquid single reagent was stored 13 months at 2-8 ℃, and the linear test result of reagent still meets the requirements.
4) stabilization AST liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 8
| 37 ℃ of shelf-times | Serum I (U/L) target value 30 (20-40) | Serum II (U/L) target value 54 (41-67) | Serum II I (U/L) target value 101 (81-121) |
| 0 day | 28 | 54 | 98 |
| 5 days | 28 | 56 | 92 |
| 6 days | 27 | 52 | 95 |
| 7 days | 26 | 53 | 95 |
Stabilization AST liquid single reagent was deposited 7 days at 37 ℃, and reagent accuracy test result is all in the target value scope that quality controlled serum indicates.
5) stabilization AST liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 9
| 2-8 ℃ of shelf-time | Serum I (U/L) | Serum II I (U/L) |
| 3 months | Target value: 28 (23-33) measured value: 32 | Target value: 104 (84-124) measured value: 117 |
| 6 months | Target value: 28 (23-33) measured value: 32 | Target value: 104 (84-124) measured value: 101 |
| 9 months | Target value: 28 (23-33) measured value: 30 | Target value: 104 (84-124) measured value: 110 |
| 12 months | Target value: 30 (20-40) measured value: 32 | Target value: 101 (81-121) measured value: 106 |
Stabilization AST liquid single reagent was deposited 12 months at 2-8 ℃, and reagent accuracy test result is all at Quality Control blood
In the clear target value scope that indicates.
Above-listed data presentation, 2-8 ℃ of this AST liquid single reagent deposit after 12 months or 37 ℃ deposited 7 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is feasible as the coenzyme reduction system of stablizing β-NADH.
Embodiment 2
The AST reagent that following mensuration is prepared according to the present invention (D-glucose: 1mmol/L, Hexose phosphate dehydrogenase: stability 5U/L):
Stabilization AST liquid single reagent prescription:
Table 10
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 100 | 12.1g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 15 | 3.39g |
| The L-aspartic acid | 133.1 | 200 | 26.6g |
| Potassium hydroxide | 56.1 | 200 | 11.2g |
| D-glucose | 180.2 | 1 | 0.18g |
| Glycerine | 92.1 | 10％ | |
| EDTA.2Na | 372.2 | 3 | 1.12g |
| Sodium azide | 65.1 | 0.3g | |
| β-NADH disodium salt | 709.4 | 0.25 | 0.18g |
| Serum lactic dehydrogenase | 1500U | ||
| Malate dehydrogenase (malic acid dehydrogenase) | 800U | ||
| Hexose phosphate dehydrogenase | 5U | ||
| Hydrochloric acid | 36.5 | Transfer pH8.0 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization AST liquid single reagent, do not contain glycerine.Other component and concentration thereof and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited.
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 15
Time of lag: 60 second test duration: 60 seconds
The reagent blank absorbancy: the content of reflection β-NADH, initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in quality controlled serum sign value scope
Test is linear: 〉=550U/L
Test result:
1) AST liquid single reagent is deposited the back blank absorbency for 37 ℃
Table 11
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.755 | 1.745 |
| 1 | 1.624 | |
| 2 | 1.490 | |
| 3 | 1.359 | 0.943 |
| 4 | 1.245 | |
| 5 | 1.110 |
2) AST liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 12
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astableization single reagent |
| 0 week | 1.758 | 1.751 |
| 3 weeks | 1.396 | |
| 5 weeks | 1.258 | |
| 7 weeks | 1.150 | |
| 11 weeks | 0.965 | |
| 3 months | 1.579 | |
| 6 months | 1.385 | |
| 9 months | 1.208 | |
| 12 months | 0.998 |
3) stabilization AST liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 13
| Deposit 3 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 31 | 125 | 250 | 374 | 499 | 624 |
| Measured value U/L | 36 | 127 | 250 | 365 | 497 | 606 |
| Deposit 6 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 30 | 122 | 243 | 365 | 486 | 608 |
| Measured value U/L | 33 | 122 | 258 | 365 | 483 | 578 |
| Deposit 9 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 30 | 110 | 220 | 330 | 440 | 550 |
| Measured value | 35 | 118 | 229 | 325 | 436 | 538 |
4) stabilization AST liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 14
| 37 ℃ of shelf-times | Serum I (U/L) target value 30 (20-40) | Serum II (U/L) target value 54 (41-67) | Serum II I (U/L) target value 101 (81-121) |
| 0 day | 33 | 56 | 107 |
| 5 days | 35 | 58 | 105 |
5) stabilization AST liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 15
| 2-8 ℃ of shelf-time | Serum I (U/L) | Serum II I (U/L) |
| 3 months | Target value: 30 (20-40) measured value: 36 | Target value: 101 (81-121) measured value: 115 |
| 6 months | Target value: 30 (20-40) measured value: 35 | Target value: 101 (81-121) measured value: 108 |
| 9 months | Target value: 30 (20-40) measured value: 32 | Target value: 101 (81-121) measured value: 106 |
Above-listed data presentation, 2-8 ℃ of this AST liquid single reagent deposit after 9 months or 37 ℃ deposited 5 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is feasible as the coenzyme reduction system of stablizing β-NADH.
Embodiment 3
The AST reagent that following mensuration is prepared according to the present invention (D-glucose: 10mmol/L, Hexose phosphate dehydrogenase: stability 50U/L):
Stabilization AST liquid single reagent prescription:
Table 16
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 90 | 10.9g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 13 | 2.9g |
| The L-aspartic acid | 133.1 | 220 | 29.3g |
| Potassium hydroxide | 56.1 | 220 | 12.3g |
| D-glucose | 180.2 | 10 | 1.8g |
| Glycerine | 92.1 | 7％ | |
| EDTA.2Na | 372.2 | 4 | 1.5g |
| Sodium azide | 65.1 | 0.4g | |
| β-NADH disodium salt | 709.4 | 0.28 | 0.199g |
| Serum lactic dehydrogenase | 1800U | ||
| Malate dehydrogenase (malic acid dehydrogenase) | 1000U | ||
| Hexose phosphate dehydrogenase | 50U | ||
| Hydrochloric acid | 36.5 | Transfer pH8.0 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization AST liquid single reagent, do not contain glycerine.Other component and concentration thereof and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited.
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 15
Time of lag: 60 second test duration: 60 seconds
The reagent blank absorbancy: the content of reflection β-NADH, initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in quality controlled serum sign value scope
Test is linear: 〉=550U/L
Test result:
1) AST liquid single reagent is deposited the back blank absorbency for 37 ℃
Table 17
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.880 | 1.886 |
| 1 | 1.759 | |
| 2 | 1.640 | |
| 3 | 1.518 | 1.075 |
| 4 | 1.396 | |
| 5 | 1.285 | |
| 6 | 1.177 | |
| 7 | 1.080 |
2) AST liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 18
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astableization single reagent |
| 0 week | 1.878 | 1.883 |
| 3 weeks | 1.530 | |
| 5 weeks | 1.395 | |
| 7 weeks | 1.283 | |
| 11 weeks | 1.092 | |
| 3 months | 1.720 | |
| 6 months | 1.541 | |
| 9 months | 1.375 | |
| 12 months | 1.192 |
3) stabilization AST liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 19
| Deposit 3 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 0 | 121.7 | 244.1 | 366.0 | 488.0 | 600 |
| Measured value U/L | 4.5 | 124 | 247 | 366 | 473 | 582 |
| Deposit 6 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 0 | 113 | 226 | 338 | 451 | 564 |
| Measured value U/L | 5.0 | 115 | 220 | 338 | 436 | 559 |
| Deposit 9 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 0 | 121 | 222 | 315 | 419 | 548 |
| Measured value U/L | 4.6 | 115 | 220 | 315 | 420 | 535 |
| Deposit 12 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 0 | 114 | 228 | 343 | 457 | 571 |
| Measured value U/L | 5.2 | 114 | 231 | 346 | 449 | 548 |
4) stabilization AST liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 20
| 2-8 ℃ of shelf-time | Serum I (U/L) | Serum II I (U/L) |
| 3 months | Target value: 28 (23-33) measured value: 31 | Target value: 104 (84-124) measured value: 105 |
| 6 months | Target value: 28 (23-33) measured value: 29 | Target value: 104 (84-124) measured value: 96 |
| 9 months | Target value: 28 (23-33) measured value: 29 | Target value: 104 (84-124) measured value: 100 |
| 12 months | Target value: 28 (23-33) measured value: 28 | Target value: 104 (84-124) measured value: 105 |
5) stabilization AST liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 21
| 37 ℃ of shelf-times | Serum I (U/L) target value 30 (20-40) | Serum II (U/L) target value 54 (41-67) | Serum II I (U/L) target value 101 (81-121) |
| 0 day | 28 | 57 | 100 |
| 5 days | 30 | 58 | 105 |
| 6 days | 29 | 56 | 104 |
| 7 days | 27 | 61 | 102 |
Above-listed data presentation, 2-8 ℃ of this AST liquid single reagent deposit after 12 months or 37 ℃ deposited 7 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is feasible as the coenzyme reduction system of stablizing β-NADH.
Embodiment 4
The ALT that following mensuration is prepared according to the present invention (D-glucose: 5mmol/L, Hexose phosphate dehydrogenase: the 10U/L) stability of reagent:
Stabilization ALT liquid single reagent:
Table 22
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 100 | 12.1g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 15 | 3.39g |
| The L-L-Ala | 89.1 | 500 | 44.6g |
| D-glucose | 180.2 | 5 | 0.9g |
| The EDTA.2 sodium salt | 372.2 | 5 | 1.86g |
| Glycerine | 92.1 | 5％ | |
| Sodium azide | 65.1 | 0.5g | |
| β-NADH disodium salt | 709.4 | 0.27 | 0.19g |
| Serum lactic dehydrogenase | 4000U | ||
| Hexose phosphate dehydrogenase | 10U | ||
| Hydrochloric acid | 36.5 | Transfer pH7.8 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization ALT liquid single reagent, do not contain glycerine.Other component concentration and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited.
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 15
Time of lag: 60 second test duration: 60 seconds
The reagent blank absorbancy: the content of reflection β-NADH, effectively initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in quality controlled serum sign value scope.
Linear: 550 U/L
Test result:
1) ALT liquid single reagent is deposited the back blank absorbency for 37 ℃
Table 23
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.857 | 1.752 |
| 1 | 1.687 | 1.375 |
| 2 | 1.066 | |
| 3 | ||
| 4 | 1.188 | |
| 5 | 1.057 |
As seen, stabilization ALT liquid single reagent can be deposited 5 days at 37 ℃, and astableization ALT liquid single reagent can only be deposited 2 days.β in the stabilization single reagent-NADH stability better.
2) ALT liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 24
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astableization single reagent |
| 0 week | 1.902 | 1.752 |
| 3 months | 1.688 | 1.235 |
| 4 months | 1.041 | |
| 6 months | 1.468 | |
| 9 months | 1.266 | |
| 12 months | 1.099 |
β-NADH can stablize more than 12 months in 2-8 ℃ of stabilization ALT liquid single reagent, but not β-NADH can only stablize four months in the stabilization single reagent.
3) stabilization ALT liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 25
| Deposit 3 months linearities for 2-8 ℃ | |||||
| Theoretical value U/L | 4.3 | 153.4 | 302.5 | 451.5 | 600.6 |
| Measured value U/L | 4.3 | 164.0 | 315.6 | 477.6 | 600.6 |
| Deposit 6 months linearities for 2-8 ℃ | |||||
| Theoretical value U/L | 8.5 | 177.0 | 345.4 | 513.9 | 682.3 |
| Measured value U/L | 8.5 | 199.1 | 367.9 | 541.4 | 682.3 |
| Deposit 8 months linearities for 2-8 ℃ | |||||
| Theoretical value U/L | 6.7 | 138.1 | 269.5 | 400.9 | 532.3 |
| Measured value U/L | 6.7 | 157.8 | 293.2 | 416.5 | 532.3 |
| Deposit 12 months linearities for 2-8 ℃ | |||||
| Theoretical value U/L | 5.9 | 154.1 | 302.3 | 450.5 | 598.7 |
| Measured value U/L | 5.9 | 169.3 | 323.1 | 471.2 | 598.7 |
Stabilization ALT liquid single reagent was stored 12 months at 2-8 ℃, and the linear test result of reagent still meets the requirements.
4) stabilization ALT liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 26
| 37 ℃ of shelf-times | Serum I (U/L) target value 23 (18-28) | Serum II (U/L) target value 45 (35-55) | Serum II I (U/L) target value 91 (76-106) |
| 0 day | 24.3 | 42.8 | 84.5 |
| 3 days | 22.1 | 41.8 | 85.6 |
| 5 days | 23.8 | 41.4 | 82.0 |
Stabilization ALT liquid single reagent was deposited 5 days at 37 ℃, and reagent accuracy test result is all in the target value scope that quality controlled serum indicates.
5) stabilization ALT liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 27
| 2-8 ℃ of shelf-time | Serum I (U/L) target value: 24 (19-29) | Serum II I (U/L) target value: 92 (77-107) |
| 3 months | Measured value: 20 | Measured value: 82 |
| 6 months | Measured value: 24 | Measured value: 78 |
| 9 months | Measured value: 23.5 | Measured value: 84 |
| 12 months | Measured value: 25.5 | Measured value: 88 |
Stabilization ALT liquid single reagent was deposited 12 months at 2-8 ℃, and reagent accuracy test result is all in the target value scope that quality controlled serum indicates.
Above-listed data presentation, 2-8 ℃ of this ALT liquid single reagent deposit after 12 months or 37 ℃ deposited 5 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is successful as the coenzyme reduction system of stablizing β-NADH.
Embodiment 5
The ALT reagent that following mensuration is prepared according to the present invention (D-glucose: 1mmol/L, Hexose phosphate dehydrogenase: stability 2U/L):
Stabilization ALT liquid single reagent prescription:
Table 28
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 80 | 9.69g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 12 | 2.71g |
| The L-L-Ala | 89.1 | 400 | 35.6g |
| D-glucose | 180.2 | 1 | 0.18g |
| The EDTA.2 sodium salt | 372.2 | 3 | 1.12g |
| Glycerine | 92.1 | 10％ | |
| Sodium azide | 65.1 | 0.3g | |
| β-NADH disodium salt | 709.4 | 0.25 | 0.177g |
| Serum lactic dehydrogenase | 3000U | ||
| Hexose phosphate dehydrogenase | 2U | ||
| Hydrochloric acid | 36.5 | Transfer pH7.8 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization ALT liquid single reagent, do not contain glycerine.Other component concentration and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited.
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 15
Time of lag: 60 second test duration: 60 seconds
The reagent blank absorbancy: the content of reflection β-NADH, effectively initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in quality controlled serum sign value scope.
Linear: 550U/L
Test result:
1) ALT liquid single reagent is deposited the back blank absorbency for 37 ℃
Table 29
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.710 | 1.705 |
| 1 | 1.525 | 1.330 |
| 2 | 1.019 | |
| 3 | ||
| 4 | 1.016 |
2) ALT liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 30
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astableization single reagent |
| 0 week | 1.715 | 1.713 |
| 3 months | 1.488 | 1.195 |
| 4 months | 1.002 | |
| 6 months | 1.250 | |
| 9 months | 1.031 |
3) stabilization ALT liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 31
| Deposit 3 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 32 | 129 | 257 | 386 | 514 | 643 |
| Measured value U/L | 30 | 128 | 257 | 376 | 507 | 629 |
| Deposit 6 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 3.9 | 104 | 208 | 312 | 417 | 520 |
| Measured value U/L | 3.9 | 117 | 220 | 312 | 404 | 513 |
| Deposit 9 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 4.6 | 117 | 234 | 351 | 468 | 565 |
| Measured value U/L | 4.6 | 112 | 237 | 351 | 459 | 536 |
4) stabilization ALT liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 32
| 2-8 ℃ of shelf-time | Serum I (U/L) target value 21 (13-29) | Serum II I (U/L) target value 93 (73-113) |
| 3 months | 28 | 90 |
| 6 months | 24 | 91 |
| 9 months | 22 | 86 |
5) stabilization ALT liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 33
| 37 ℃ of shelf-times | Serum I (U/L) target value 24 (19-29) | Serum II (U/L) target value 47 (37-57) | Serum II I (U/L) target value 92 (77-107) |
| 0 day | 22 | 51 | 90 |
| 4 days | 28 | 46 | 87 |
Above-listed data presentation, 2-8 ℃ of this ALT liquid single reagent deposit after 9 months or 37 ℃ deposited 4 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is successful as the coenzyme reduction system of stablizing β-NADH.
Embodiment 6
The ALT reagent that following mensuration is prepared according to the present invention (D-glucose: 10mmol/L, Hexose phosphate dehydrogenase: stability 50U/L):
Stabilization ALT liquid single reagent prescription:
Table 34
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 90 | 10.9g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 13 | 2.94g |
| The L-L-Ala | 89.1 | 450 | 40.1g |
| D-glucose | 180.2 | 10 | 1.8g |
| The EDTA.2 sodium salt | 372.2 | 4 | 1.49g |
| Glycerine | 92.1 | 7％ | |
| Sodium azide | 65.1 | 0.4g | |
| β-NADH disodium salt | 709.4 | 0.28 | 0.199g |
| Serum lactic dehydrogenase | 3500U | ||
| Hexose phosphate dehydrogenase | 50U | ||
| Hydrochloric acid | 36.5 | Transfer pH7.8 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization ALT liquid single reagent, do not contain glycerine.Other component concentration and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited.
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 15
Time of lag: 60 second test duration: 60 seconds
The reagent blank absorbancy: the content of reflection β-NADH, effectively initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in quality controlled serum sign value scope.
Linear: 〉=550U/L
Test result:
1) ALT liquid single reagent is deposited the back blank absorbency for 37 ℃
Table 35
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.881 | 1.875 |
| 1 | 1.715 | 1.493 |
| 2 | 1.154 | |
| 3 | ||
| 4 | 1.220 | |
| 5 | 1.092 |
2) ALT liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 36
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astable listization reagent |
| 0 week | 1.915 | 1.880 |
| 3 months | 1.697 | 1.365 |
| 4 months | 1.169 | |
| 6 months | 1.478 | |
| 9 months | 1.280 | |
| 12 months | 1.115 |
3) stabilization ALT liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 37
| Deposit 3 months linearities for 2-8 ℃ | |||||||||
| Theoretical value U/L | 5.1 | 142 | 284 | 425 | 567 | ||||
| Measured value U/L | 5.1 | 142 | 294 | 412 | 555 | ||||
| Deposit 6 months linearities for 2-8 ℃ | |||||||||
| Theoretical value U/L | 4.5 | 125 | 246 | 369 | 492 | 615 | |||
| Measured value U/L | 4.5 | 121 | 244 | 372 | 487 | 604 | |||
| Deposit 9 months linearities for 2-8 ℃ | |||||||||
| Theoretical value U/L | 5.5 | 80 | 160 | 321 | 481 | 641 |
| Measured value U/L | 5.5 | 85 | 170 | 321 | 473 | 625 |
| Deposit 12 months linearities for 2-8 ℃ | ||||||
| Theoretical value U/L | 98 | 195 | 293 | 390 | 488 | 580 |
| Measured value U/L | 100 | 199 | 287 | 398 | 479 | 561 |
4) stabilization ALT liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 38
| 2-8 ℃ of shelf-time | Serum I (U/L) target value 21 (13-29) | Serum II I (U/L) target value 93 (73-113) |
| 3 months | 23 | 84 |
| 6 months | 21 | 86 |
| 9 months | 26 | 82 |
| 12 months | 22 | 87 |
5) stabilization ALT liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 39
| 37 ℃ of shelf-times | Serum I (U/L) target value 24 (1 9-29) | Serum II (U/L) target value 47 (37-57) | Serum II I (U/L) target value 92 (7%107) |
| 0 day | 21 | 45 | 86 |
| 5 days | 23 | 44 | 84 |
Above-listed data presentation, 2-8 ℃ of this ALT liquid single reagent deposit after 12 months or 37 ℃ deposited 5 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is successful as the coenzyme reduction system of stablizing β-NADH.
Embodiment 7
The UREA reagent that following mensuration is prepared according to the present invention (D-glucose: 5mmol/L, Hexose phosphate dehydrogenase: stability 30U/L):
Stabilization UREA liquid single reagent:
Table 40
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 100 | 12.1g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 7 | 1.6g |
| Sodium azide | 65.0 | 0.3g | |
| D-glucose | 180.2 | 5 | 0.9g |
| Glycerine | 92.1 | 10％ | |
| ADP.K salt | 501.3 | 2 | 1.0g |
| β-NADH, sodium salt | 709.4 | 0.28 | 0.2g |
| Glutamate dehydrogenase | 600U/L | ||
| Urase | 6000U/L | ||
| Hexose phosphate dehydrogenase | 30U/L | ||
| Hydrochloric acid | 36.5 | Transfer pH8.1 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization UREA liquid single reagent, do not contain glycerine.Other component concentration and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 100
Time of lag: 30 second test duration: 60-150 second
The reagent blank absorbancy: the content of reflection β-NADH, effectively initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in sign be worth scope
Linear: 〉=50mmol/L
Test result:
1) Urea liquid single reagent is deposited the back blank absorbency for 37 ℃
Table 41
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.821 | 1.835 |
| 1 | 1.655 | 1.621 |
| 2 | 1.547 | 1.424 |
| 3 | 1.421 | 1.267 |
| 4 | 1.302 | 1.113 |
| 5 | 1.200 | 0.956 |
| 6 | 1.126 | |
| 7 | 1.051 |
As seen, stabilization Urea liquid single reagent can be deposited 7 days at 37 ℃, and astableization BUN liquid single reagent can only be deposited 4 days.β in the stabilization single reagent-NADH stability better.
2) Urea liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 42
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astableization single reagent |
| 0 month | 1.773 | 1.835 |
| 3 months | 1.611 | 1.519 |
| 6 months | 1.507 | 1.271 |
| 8 months | 1.035 | |
| 9 months | 1.391 | |
| 12 months | 1.226 | |
| 15 months | 1.125 | |
| 18 months | 1.029 |
β-NADH can stablize more than 18 months in 2-8 ℃ of stabilization Urea liquid single reagent, but not β-NADH can only stablize 8 months in the stabilization Urea liquid single reagent.
3) stabilization Urea liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 43
| Deposit 4 months linearities for 2-8 ℃ | |||||||||||
| Theoretical value (mmol/l) | 1.59 | 10.58 | 21.16 | 31.74 | 42.32 | 52.90 | |||||
| Measured value (mmol/L) | 1.92 | 11.18 | 22.21 | 31.74 | 43.69 | 52.89 | |||||
| Deposit 6 months linearities for 2-8 ℃ | |||||||||||
| Theoretical value (mmol/l) | 1.68 | 14.00 | 28.00 | 42.00 | 56.00 | ||||||
| Measured value (mmol/L) | 1.82 | 14.32 | 28.09 | 41.95 | 54.26 | ||||||
| Deposit 9 months linearities for 2-8 ℃ | |||||||||||
| Theoretical value (mmol/l) | 1.62 | 13.50 | 27.00 | 40.50 | 54.00 | ||||||
| Measured value (mmol/L) | 1.80 | 14.14 | 27.55 | 40.16 | 52.08 | ||||||
| Deposit 12 months linearities for 2-8 ℃ | |||||||||||
| Theoretical value (mmol/l) | 1.62 | 13.50 | 27.00 | 40.50 | 54.00 |
| Measured value (mmol/L) | 1.74 | 14.07 | 27.86 | 39.67 | 51.70 |
| Deposit 15 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/l) | 1.62 | 13.50 | 27.00 | 40.50 | 54.00 |
| Measured value (mmol/L) | 1.80 | 13.39 | 27.00 | 38.12 | 50.15 |
| Deposit 18 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/l) | 1.68 | 14.00 | 28.00 | 42.00 | 56.00 |
| Measured value (mmol/L) | 1.80 | 14.59 | 28.44 | 40.61 | 53.38 |
Stabilization Urea liquid single reagent was stored 18 months at 2-8 ℃, and the linear test result of reagent still meets the requirements.
4) stabilization Urea liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 44
| 37 ℃ of shelf-times | Serum I (mmol/L) target value 3.0 (2.3-3.7) | Serum II (mmol/L) target value 10.2 (8.7-11.7) | Serum II I (mmol/L) target value 18.7 (16.5-20.8) |
| 0 day | 3.1 | 10.4 | 18.8 |
| 5 days | 3.12 | 10.08 | 19.54 |
| 7 days | 2.97 | 10.11 | 18.77 |
Stabilization Urea liquid single reagent was deposited 7 days at 37 ℃, and reagent accuracy test result is all in the target value scope that quality controlled serum indicates.
5) stabilization Urea liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 45
| 2-8 ℃ of shelf-time | Serum I (mmol/L) | Serum II I (mmol/L) |
| 3 months | Target value 2.5 (1.8-3.2) measured value 2.76 | Target value 18.5 (16.3-20.7) measured value 18.06 |
| 6 months | Target value 2.5 (1.8-3.2) measured value 2.52 | Target value 18.5 (16.3-20.7) measured value 19.40 |
| 9 months | Target value 2.5 (1.8-3.2) measured value 2.72 | Target value 18.5 (16.3-20.7) measured value 19.05 |
| 12 months | Target value 7.70 (5.58-9.55) measured value 7.86 | |
| 15 months | Target value 3.0 (2.3-3.7) measured value 2.83 | Target value 18.7 (16.5-20.8) measured value 17.27 |
| 18 months | Target value 3.0 (2.3-3.7) measured value 2.95 | Target value 18.7 (16.5-20.8) measured value 19.05 |
Stabilization Urea liquid single reagent was deposited 12 months at 2-8 ℃, and reagent accuracy test result is all in the target value scope that serum indicates.
Above-listed data presentation, 2-8 ℃ of Urea liquid single reagent deposit after 18 months or 37 ℃ deposited 7 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is feasible as the coenzyme reduction system of stablizing β-NADH.
Embodiment 8
The UREA reagent that following mensuration is prepared according to the present invention (D-glucose: 1mmol/L, Hexose phosphate dehydrogenase: stability 5U/L):
Stabilization UREA liquid single reagent prescription:
Table 46
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 110 | 13.3g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 4 | 9.04g |
| Sodium azide | 65.0 | 0.2g | |
| D-glucose | 180.2 | 1 | 0.18g |
| Glycerine | 92.1 | 5％ | |
| ADP.K salt | 501.3 | 1 | 0.5g |
| β-NADH, sodium salt | 709.4 | 0.25 | 0.177g |
| Glutamate dehydrogenase | 500U | ||
| Urase | 5000U | ||
| Hexose phosphate dehydrogenase | 5U | ||
| Hydrochloric acid | 36.5 | Transfer pH8.1 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization UREA liquid single reagent, do not contain glycerine.Other component concentration and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 100
Time of lag: 30 second test duration: 60-150 second
The reagent blank absorbancy: the content of reflection β-NADH, effectively initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in sign be worth scope
Linear: 〉=50mmol/L
1) Urea liquid single reagent is deposited the back blank absorbency for 37 ℃
Table 47
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.550 | 1.557 |
| 1 | 1.379 | 1.345 |
| 2 | 1.265 | 1.144 |
| 3 | 1.130 | 0.986 |
| 4 | 1.008 |
2) Urea liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 48
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astableization single reagent |
| 0 month | 1.562 | 1.555 |
| 3 months | 1.393 | 1.240 |
| 6 months | 1.285 | 0.996 |
| 9 months | 1.163 | |
| 12 months | 1.001 |
3) stabilization Urea liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 49
| Deposit 3 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 1.70 | 12.80 | 25.60 | 38.40 | 51.20 |
| Measured value (mmol/L) | 1.80 | 13.39 | 27.00 | 38.12 | 50.15 |
| Deposit 6 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 10.50 | 21.00 | 31.50 | 42.00 | 52.50 |
| Measured value (mmol/L) | 11.36 | 22.11 | 31.75 | 41.61 | 52.29 |
| Deposit 9 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 10.50 | 21.00 | 31.50 | 42.00 | 52.50 |
| Measured value (mmol/L) | 11.12 | 21.84 | 31.61 | 41.50 | 50.18 |
| Deposit 12 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 10.22 | 20.44 | 30.66 | 40.88 | 51.10 |
| Measured value (mmol/L) | 10.80 | 20.50 | 29.77 | 39.14 | 48.75 |
4) stabilization Urea liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 50
| 37 ℃ of shelf-times | Serum I (mmol/L) target value 2.5 (1.8-3.2) | Serum II (mmol/L) target value 9.0 (7.5-10.5) | Serum II I (mmol/L) target value 18.5 (16.3-20.7) |
| 0 day | 2.67 | 9.83 | 18.14 |
| 4 days | 2.85 | 9.77 | 18.50 |
5) stabilization Urea liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 51
| 2-8 ℃ of shelf-time | Serum I (mmol/L) target value 2.7 (2.0-3.3) | Serum II I (mmol/L) target value 18.7 (16.5-20.8) |
| 3 months | 2.91 | 19.20 |
| 6 months | 3.00 | 19.01 |
| 9 months | 2.98 | 18.90 |
| 12 months | 2.90 | 19.00 |
Above-listed data presentation, 2-8 ℃ of this Urea liquid single reagent deposit after 12 months or 37 ℃ deposited 4 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is feasible as the coenzyme reduction system of stablizing β-NADH.
Embodiment 9
The UREA reagent that following mensuration is prepared according to the present invention (D-glucose: 10mmol/L, Hexose phosphate dehydrogenase: stability 50U/L):
Stabilization UREA liquid single reagent prescription:
Table 52
| Raw material | Molecular weight | Concentration (mmol/L) | Every liter of amount |
| Tris | 121.1 | 120 | 14.5g |
| α-Tong Wuersuan, Na salt (2H 2O) | 226.1 | 8 | 2.26g |
| Sodium azide | 65.0 | 0.5g | |
| D-glucose | 180.2 | 10 | 1.8g |
| Glycerine | 92.1 | 15％ | |
| ADP.K salt | 501.3 | 4 | 2g |
| β-NADH, sodium salt | 709.4 | 0.3 | 0.21g |
| Glutamate dehydrogenase | 1000U | ||
| Urase | 8000U | ||
| Hexose phosphate dehydrogenase | 50U | ||
| Hydrochloric acid | 36.5 | Transfer pH8.1 |
Do not contain Hexose phosphate dehydrogenase/D-glucose coenzyme reduction system in the corresponding astableization UREA liquid single reagent, do not contain glycerine.Other component concentration and last epiphase are together.
Reagent storage requirement: a 2-8 ℃ sealing is deposited, and 37 ℃ of sealings are deposited
Test wavelength: 340nm probe temperature: 37 ℃
The cuvette optical path: the 10mm sample compares with reagent volume: 1: 100
Time of lag: 30 second test duration: 60-150 second
The reagent blank absorbancy: the content of reflection β-NADH, effectively initial absorbance should be greater than 1.0A
Accuracy: measurement result should be in sign be worth scope
Linear: 〉=50mmol/L
1) UREA liquid single reagent is deposited the back blank absorbency for 37 ℃
53
| Deposit fate for 37 ℃ | The stabilization single reagent | Astableization single reagent |
| 0 | 1.942 | 1.938 |
| 1 | 1.780 | 1.723 |
| 2 | 1.675 | 1.520 |
| 3 | 1.553 | 1.362 |
| 4 | 1.440 | 1.205 |
| 5 | 1.341 | 1.047 |
| 6 | 1.273 | |
| 7 | 1.205 |
2) Urea liquid single reagent is deposited the back blank absorbency for 2-8 ℃
Table 54
| 2-8 ℃ of shelf-time | The stabilization single reagent | Astableization single reagent |
| 0 month | 1.933 | 1.930 |
| 3 months | 1.770 | 1.613 |
| 6 months | 1.669 | 1.362 |
| 8 months | 1.124 | |
| 9 months | 1.555 | |
| 12 months | 1.392 | |
| 15 months | 1.295 | |
| 18 months | 1.203 |
3) stabilization Urea liquid single reagent is deposited the back linear determination for 2-8 ℃
Table 55
| Deposit 3 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 11.47 | 22.93 | 34.40 | 45.87 | 57.34 |
| Measured value (mmol/L) | 11.96 | 23.02 | 34.40 | 43.62 | 55.43 |
| Deposit 6 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 10.03 | 20.06 | 30.09 | 40.12 | 50.15 |
| Measured value (mmol/L) | 10.03 | 20.80 | 31.43 | 40.12 | 50.29 |
| Deposit 9 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 11.30 | 22.60 | 34.00 | 45.30 | 56.60 |
| Measured value (mmol/L) | 11.90 | 23.20 | 34.00 | 43.70 | 54.20 |
| Deposit 12 months linearities for 2-8 ℃ | |||||
| Theoretical value (mrnol/L) | 11.66 | 23.31 | 34.97 | 46.63 | 58.29 |
| Measured value (mmol/L) | 12.07 | 24.44 | 34.97 | 46.22 | 56.96 |
| Deposit 15 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 11.00 | 21.90 | 32.90 | 43.80 | 54.80 |
| Measured value (mmol/L) | 11.80 | 22.40 | 31.70 | 43.80 | 52.30 |
| Deposit 18 months linearities for 2-8 ℃ | |||||
| Theoretical value (mmol/L) | 10.27 | 20.54 | 30.80 | 41.07 | 51.34 |
| Measured value (mmol/L) | 11.09 | 21.73 | 31.95 | 41.07 | 50.81 |
4) stabilization Urea liquid single reagent is deposited the back accuracy determination for 37 ℃
Table 56
| 37 ℃ of shelf-times | Serum I (mmol/L) target value 2.5 (1.8-3.2) | Serum II (mmol/L) target value 9.0 (7.5-10.5) | Serum II I (mmol/L) target value 18.5 (16.3-20.7) |
| 0 day | 2.64 | 9.17 | 18.64 |
| 4 days | 2.51 | 9.70 | 18.73 |
| 7 days | 2.47 | 9.64 | 19.20 |
5) stabilization Urea liquid single reagent is deposited the back accuracy determination for 2-8 ℃
Table 57
| 2-8 ℃ of shelf-time | Serum I (mmol/L) target value 2.7 (2.0-3.3) | Serum II I (mmol/L) target value 18.7 (16.5-20.8) |
| 3 months | 2.77 | 18.35 |
| 6 months | 2.80 | 18.86 |
| 9 months | 2.90 | 18.65 |
| 12 months | 2.92 | 18.60 |
| 15 months | 2.98 | 19.0 |
| 18 months | 3.00 | 18.73 |
Above-listed data presentation, 2-8 ℃ of Urea liquid single reagent deposit after 18 months or 37 ℃ deposited 7 days, the reagent test result is normal.Using highly narrow spectrum Hexose phosphate dehydrogenase and D-glucose is feasible as the coenzyme reduction system of stablizing β-NADH.
Industrial applicibility
The present invention has used highly narrow spectrum enzyme/substrate pair owing to be used for stable reagent with oxidation resistant coenzyme reduction system, the consumption of enzyme and substrate greatly reduces, not only increase hardly reagent cost, and can not introduce new assorted enzyme because of the adding of a large amount of stabilized enzyme, thereby improved the stability of reagent.
Claims (11)
1, a kind of method of enzymatic assays patient analyte concentration, it is characterized in that: during mensuration the rate of oxidation of measuring reduced coenzyme in the used reagent is measured, dynamic stability by the cyclic regeneration reduced coenzyme in Hexose phosphate dehydrogenase/D-glucose is used for realizing that this reagent is 2-8 ℃ of lay up period long-term stability, in described Hexose phosphate dehydrogenase/D-glucose, Hexose phosphate dehydrogenase has specificity completely to D-glucose.
2, the method for enzymatic assays patient analyte concentration according to claim 1 is characterized in that: described reagent is the liquid single reagent.
3, the method for enzymatic assays patient analyte concentration according to claim 1 and 2, it is characterized in that: wherein said analyte is an aspartic transaminase.
4, the method for enzymatic assays patient analyte concentration according to claim 3 is characterized in that: described Hexose phosphate dehydrogenase consumption is 2-100U/L, and described D-glucose consumption is 0.1-20mmol/L.
5, the method for enzymatic assays patient analyte concentration according to claim 4 is characterized in that: described Hexose phosphate dehydrogenase consumption is 5-50U/L, and described D-glucose consumption is 1-10mmol/L.
6, the method for enzymatic assays patient analyte concentration according to claim 1 and 2, it is characterized in that: described analyte is an alanine aminotransferase.
7, the method for enzymatic assays patient analyte concentration according to claim 6 is characterized in that: described Hexose phosphate dehydrogenase consumption is 2-100U/L, and described D-glucose consumption is 0.1-20mmol/L.
8, the method for enzymatic assays patient analyte concentration according to claim 7 is characterized in that: described Hexose phosphate dehydrogenase consumption is 2-50U/L, and described D-glucose consumption is 1-10mmol/L.
9, the method for enzymatic assays patient analyte concentration according to claim 1 and 2, it is characterized in that: described analyte is a blood urea.
10, the method for enzymatic assays patient analyte concentration according to claim 9 is characterized in that: described Hexose phosphate dehydrogenase consumption is 2-100U/L, and described D-glucose consumption is 0.1-20mmol/L.
11, the method for enzymatic assays patient analyte concentration according to claim 10 is characterized in that: described Hexose phosphate dehydrogenase consumption is 5-50U/L, and described D-glucose consumption is 1-10mmol/L.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2003/000749 WO2005024014A1 (en) | 2003-09-05 | 2003-09-05 | The agent for assaying analyte of patient by enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1820071A CN1820071A (en) | 2006-08-16 |
| CN100359008C true CN100359008C (en) | 2008-01-02 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB038269554A Expired - Fee Related CN100359008C (en) | 2003-09-05 | 2003-09-05 | Reagent for determining analyte concentration of patient by enzyme method |
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| Country | Link |
|---|---|
| US (1) | US20070048813A1 (en) |
| CN (1) | CN100359008C (en) |
| AU (1) | AU2003261614A1 (en) |
| WO (1) | WO2005024014A1 (en) |
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| CN104404127B (en) * | 2014-11-28 | 2017-06-06 | 山东博科生物产业有限公司 | A kind of strong blood Detection reagent for alanine aminotransferase of stability |
| CN106885905A (en) * | 2015-12-16 | 2017-06-23 | 山东博科生物产业有限公司 | A kind of urea detection reagent and detection method with superior detection line and sensitivity for analysis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4394449A (en) * | 1980-02-13 | 1983-07-19 | Modrovich Ivan Endre | Stabilization of coenzymes in aqueous solution |
| CN1133070A (en) * | 1993-09-17 | 1996-10-09 | 微量科学有限公司 | Reagent |
| CN1179792A (en) * | 1995-03-28 | 1998-04-22 | 微量科学有限公司 | Reagent stabilized using coenzyme reduction system |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6306594B1 (en) * | 1988-11-14 | 2001-10-23 | I-Stat Corporation | Methods for microdispensing patterened layers |
-
2003
- 2003-09-05 US US10/574,643 patent/US20070048813A1/en not_active Abandoned
- 2003-09-05 CN CNB038269554A patent/CN100359008C/en not_active Expired - Fee Related
- 2003-09-05 WO PCT/CN2003/000749 patent/WO2005024014A1/en active Application Filing
- 2003-09-05 AU AU2003261614A patent/AU2003261614A1/en not_active Abandoned
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4394449A (en) * | 1980-02-13 | 1983-07-19 | Modrovich Ivan Endre | Stabilization of coenzymes in aqueous solution |
| CN1133070A (en) * | 1993-09-17 | 1996-10-09 | 微量科学有限公司 | Reagent |
| CN1179792A (en) * | 1995-03-28 | 1998-04-22 | 微量科学有限公司 | Reagent stabilized using coenzyme reduction system |
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| Publication number | Publication date |
|---|---|
| AU2003261614A1 (en) | 2005-03-29 |
| CN1820071A (en) | 2006-08-16 |
| WO2005024014A1 (en) | 2005-03-17 |
| US20070048813A1 (en) | 2007-03-01 |
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